Project Summary: Systemic listeriosis is one of the most deadly foodborne bacterial diseases in North America, as evidenced by recent outbreaks caused by contaminated cantaloupes (2011; 21% mortality) and cold cuts (2008; 39% mortality). After Listeria monocytogenes (Lm) spread to the brain, it is difficult to eradicate the infection, and many patients die even while receiving aggressive antibiotic treatment. Two different types of brain infections are observed in humans: a diffuse meningitis/meningoencephalitis typically seen in the elderly and other immune-compromised individuals; and a more focal infection localized in the brainstem (rhombencephalitis) that occurs in otherwise healthy adults. The central hypothesis of this proposal is that meningitis occurs via hematogenous spread, primarily due to a failure of the immune system to limit overwhelming growth of Lm in peripheral tissues, while rhombencephalitis occurs when neurotropic strains of Lm invade cranial nerves and spread to the brain by axonal migration. Progress in understanding brain infections has been limited by the lack of an appropriate small animal model to study the two proposed routes of dissemination, and by the technical limitation that very few bacteria are needed to cause symptomatic disease in the brain. In Aim 1 of this proposal, bacterial colonization patterns in the brain (focal vs. diffuse) will be identified in mice given food contaminated with either neurotropic Lm (human rhombencephalitis and sheep brain isolates) or non-neurotropic Lm (veterinary strains isolated from the liver). In Aim 2, an innovative approach will define the pathway used by neurovirulent Lm to disseminate to the brain. Lm will be engineered to secrete Cre recombinase during intracellular growth, and then used to infect mice with a ?floxed? GFP gene, allowing for permanent identification of all cells in the body that harbored intracellular Lm during any point of the infection. A key strength of this proposal is the interdisciplinary approach resulting from a collaboration between microbiologists in the D?Orazio lab with extensive expertise in manipulating Lm and studying host responses to infection, and neuroscientists in the Gensel lab, who have the expertise to perform high resolution imaging of the nerves and brainstems of mice. Working together, this group will advance the field by revealing how Lm disseminate to the brain following natural foodborne transmission, and will develop a set of tools that may be used in future studies to identify the features of neurotropic Lm strains that promote brain infection.